We have identified and cloned a cDNA encoding a novel splice variant of the SDF1 gene, SDF-1gamma. SDF-1gamma contains a short domain highly homologous to the RNA binding domain of HIV-Tat. We hypothesized that SDF-1gamma inhibits Tat- dependent HIV transcription and suppress HIV replication by competing with Tat for TAR binding. Preliminary experiments indicate that SDF-1gamma indeed inhibits Tat-dependent HIV transcription. The major goal of this proposal is to focus on endogenous SDF-1gamma and to study its role in HIV biology. We propose to: 1. To quantitatively measure SDF-1y expression at the RNA and protein levels and to obtain biologically active recombinant protein. We will develop reagents to quantitatively detect SDF-1gamma mRNA (real-time TaqMan PCR) and protein (specific antiserum). We will examine SDF-1gamma levels in different cell types in response to immune activation signals and HIV infection. We will express recombinant SDF-1gamma in Escherichia coli and in mammalian cells, purify it, and test its biological activities. 2. To determine the role of endogenous SDF-1gamma in HIV transcription and replication. We will use the RNA interference approach to selectively knockdown SDF-1gamma expression. We will introduce an shRNA specific for SDF-1y using a retroviral vector in lymphoid cells. HIV transcription and replication will be assessed in the presence and absence of SDF-1gamma. 3. To explore the mechanism of action of SDF-1gamma on Tat-mediated transactivation. Our preliminary experiments indicate that SDF-1gamma binds to the Tat responsive element TAR. We will test whether SDF-1gamma competes for binding with the transactivator Tat. We will perform a mutational analysis of the SDF-1gamma open reading frame to determine which domains are critical for the inhibition of HIV transactivation. 4. To assess the role of the SDF1-3'A gene polymorphism in SDF-1gamma expression and in HIV replication in vitro. A unique polymorphism in the SDF1 gene has been linked to accelerated progression to AIDS in HIV-infected patients. This polymorphism is present in the SDF-1gamma messenger RNA and could affect the expression levels of SDF-1gamma. We will measure mRNA and protein levels for SDF-1alpha, beta, and gamma in lysates and supernatants from lymphocyte culture of normal individuals carrying none, one or two copies of the SDF1-3'A allele. We will isolate primary lymphocytes from these normal donors and measure HIV gene expression in their cells using a one-step growth curve. We will determine whether a correlations exist between SDF-1gamma expression, HIV replication and the presence of the SDF1-3'A allele.